CpG-ODN attenuates pathological cardiac hypertrophy and heart failure by activation of PI3Kα-Akt signaling.

Phosphoinositide-3-kinase α (PI3Kα) represents a potential novel drug target for pathological cardiac hypertrophy (PCH) and heart failure. Oligodeoxynucleotides containing CpG motifs (CpG-ODN) are classic agonists of Toll-like receptor 9 (TLR9), which typically activates PI3K-Akt signaling in immune cells; however, the role of the nucleotide TLR9 agonists in cardiac myocytes is largely unknown. Here we report that CpG-ODN C274 could both attenuate PCH and improve cardiac dysfunction by activating PI3Kα-Akt signaling cascade. In vitro studies indicated that C274 could blunt reactivation of fetal cardiac genes and cell enlargement induced by a hypertrophic agent, isoproterenol. The anti-hypertrophic effect of C274 was suppressed by a pan-PI3K inhibitor, LY294002, or a small interfering RNA targeting PI3Kα. In vivo studies demonstrated that PCH, as marked by increased heart weight (HW) and cardiac ANF mRNA, was normalized by pre-administration with C274. In addition, Doppler echocardiography detected cardiac ventricular dilation, and contractile dysfunction in isoproterenol-treated animals, consistent with massive replacement fibrosis, reflecting cardiac cell death. As expected, pre-treatment of mice with C274 could prevent cardiac dysfunction associated with diminished cardiac cell death and fibrosis. In conclusion, CpG-ODNs are novel cardioprotective agents possessing antihypertrophic and anti-cell death activity afforded by engagement of the PI3Kα-Akt signaling. CpG-ODNs may have clinical use curbing the progression of PCH and preventing heart failure.

Hypoxia-induced acidosis uncouples the STIM-Orai calcium signaling complex.

The endoplasmic reticulum Ca(2+)-sensing STIM proteins mediate Ca(2+) entry signals by coupling to activate plasma membrane Orai channels. We reveal that STIM-Orai coupling is rapidly blocked by hypoxia and the ensuing decrease in cytosolic pH. In smooth muscle cells or HEK293 cells coexpressing STIM1 and Orai1, acute hypoxic conditions rapidly blocked store-operated Ca(2+) entry and the Orai1-mediated Ca(2+) release-activated Ca(2+) current (I(CRAC)). Hypoxia-induced blockade of Ca(2+) entry and I(CRAC) was reversed by NH(4)(+)-induced cytosolic alkalinization. Hypoxia and acidification both blocked I(CRAC) induced by the short STIM1 Orai-activating region. Although hypoxia induced STIM1 translocation into junctions, it did not dissociate the STIM1-Orai1 complex. However, both hypoxia and cytosolic acidosis rapidly decreased Förster resonance energy transfer (FRET) between STIM1-YFP and Orai1-CFP. Thus, although hypoxia promotes STIM1 junctional accumulation, the ensuing acidification functionally uncouples the STIM1-Orai1 complex providing an important mechanism protecting cells from Ca(2+) overload under hypoxic stress conditions.

Protein kinase D3 is a pivotal activator of pathological cardiac hypertrophy by selectively increasing the expression of hypertrophic transcription factors.

Fetal cardiac gene reactivation is a hallmark of pathological cardiac hypertrophy (PCH) driven by cardiac transcription factors (TFs) such as nuclear factor of activated T-cells (NFATs). Nuclear import of dephosphorylated NFATs catalyzed by calcineurin (CaN) is a well-established hypertrophic mechanism. Here we report that NFATc4 expression is also up-regulated by newly expressed protein kinase D3 (PKD3) to induce PCH. In both in vitro and in vivo cardiac hypertrophic models, the normally undetectable PKD3 was profoundly up-regulated by isoproterenol followed by overt expression of cardiac TFs including NFATc4, NK family of transcription factor 2.5 (Nkx2.5), GATA4 and myocyte enhancer factor 2 (MEF2). Using gene silencing approaches, we demonstrate PKD3 is required for increasing the expression of NFATc4, Nkx2.5, and GATA4 while PKD1 is required for the increase in MEF2D expression. Upstream induction of PKD3 is driven by nuclear entry of CaN-activated NFATc1 and c3 but not c4. Therefore, PKD3 is a pivotal mediator of the CaN-NFATc1/c3-PKD3-NFATc4 hypertrophic signaling cascade and a potential new drug target for the PCH.

Different effects of epidermal growth factor on smooth muscle cells derived from human myometrium and from leiomyoma.

OBJECTIVE: To determine different effects of epidermal growth factor (EGF) on cultured smooth muscle cells (SMCs) derived from human myometrium and leiomyoma. DESIGN: EGF effects on DNA synthesis and intracellular signal transduction were studied in cultured SMCs from leiomyoma and its matched myometrium. SETTING: Research laboratories. PATIENT(S): Patients 35-50 years old with uterine leiomyomas. INTERVENTION(S): Hysterectomy. MAIN OUTCOME MEASURE(S): Signal transduction from EGF receptor. RESULT(S): As analyzed by laser scanning cytometry (LSC), EGF treatment stimulated DNA synthesis and induced polyploidization of leiomyomal, but not myometrial, SMCs. EGF stimulation was inhibited by AG1478, an EGF receptor (EGFR) inhibitor and PD98059, a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor. Both leiomyomal and myometrial SMCs had similar expression levels of EGFR, but EGF treatment induced transient phosphorylation activation of EGFR and Akt in leiomyomal SMCs. Consequently, EGF triggered transient phosphorylation activation of p44/42 MAPK in leiomyomal SMCs, followed by down-regulation of p27. In myometrial SMCs, however, EGF induced sustained activation of EGFR, Akt, and p44/42 MAPK with up-regulation of p27. CONCLUSION(S): EGF stimulates DNA synthesis and polyploidization in leiomyomal SMCs through transient activation of the EGFR-MAPK pathway. Given that polyploidization plays a role in tumorigenesis, our results shed new light on the pathogenesis of human uterine leiomyoma.

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a new promising target for prevention and treatment of cardiac hypertrophy and heart failure. There are three δ isoforms of CaMKII in the heart and previous studies focused primarily on δB and δC types. Here we report the δA isoform of CaMKII is also critically involved in cardiac hypertrophy. We found that δA was significantly upregulated in pathological cardiac hypertrophy in both neonatal and adult models. Upregulation of δA was accompanied by cell enlargement, sarcomere reorganization and reactivation of various hypertrophic cardiac genes including atrial natriuretic factor (ANF) and ß-myocin heavy chain (ß-MHC). Studies further indicated the pathological changes were largely blunted by silencing the δA gene and an underlying mechanism indicated selective interference with the HDAC4-MEF2 signaling pathway. These results provide new evidence for selective interfering cardiac hypertrophy and heart failure when CaMKII is considered as a therapeutic target.

Rapamycin inhibits hydrogen peroxide-induced loss of vascular contractility.

Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, has been shown to extend the life span of mice, and oxidative stress plays critical roles in vascular aging involving loss of compliance of arteries. We examined, therefore, whether rapamycin has protective effects on the inhibition of vascular contractility by hydrogen peroxide (H2O2). Prolonged (3 h) exposure to H2O2 induced complete loss of contraction of mouse aortic rings and mesenteric (resistance) arteries to either KCl or phenylephrine, which was attenuated by pretreatment with rapamycin. H2O2-induced loss of contractility was unaffected by treatment with actinomycin D or cycloheximide, inhibitors of gene transcription and protein synthesis, respectively. Western blot analysis showed that there was no increase in phosphorylation of S6 kinase 1 (S6K) or factor 4E binding protein 1 (4EBP1) in response to H2O2 treatment, suggesting involvement of the mTOR complex-2 (mTORC2) rather than mTORC1. H2O2 treatment inhibited phosphorylation of the 20-kDa regulatory light chains of myosin (LC20), which was partially blocked by rapamycin treatment. Interestingly, the calcineurin inhibitors cyclosporine A and FK506 were found to mimic the rapamycin effect, and rapamycin inhibited calcineurin activation induced by H2O2. We conclude that rapamycin inhibits H2O2-induced loss of vascular contractility, likely through an mTORC2-calcineurin pathway.

Increased expression of tuberin in human uterine leiomyoma.

Female Eker rats harboring an insertional deletion in one copy of the tuberous sclerosis complex 2 (Tsc2) gene develop uterine leiomyoma, but the underlying mechanism of human uterine leiomyoma is not completely understood. To examine whether down-regulation of tuberin, a TSC2 gene product, is present in human uterine leiomyoma, we analyzed leiomyoma and matched myometrium tissues from 22 Chinese patients with Western blotting and real-time polymerase chain reaction analyses, and found that the expression of tuberin was significantly increased in leiomyoma tissues compared with matched myometrium tissues with inhibition of both the mammalian target of rapacmycin pathway and mitogen-activated protein kinase pathways.

The calcium store sensor, STIM1, reciprocally controls Orai and CaV1.2 channels.

Calcium signals, pivotal in controlling cell function, can be generated by calcium entry channels activated by plasma membrane depolarization or depletion of internal calcium stores. We reveal a regulatory link between these two channel subtypes mediated by the ubiquitous calcium-sensing STIM proteins. STIM1 activation by store depletion or mutational modification strongly suppresses voltage-operated calcium (Ca(V)1.2) channels while activating store-operated Orai channels. Both actions are mediated by the short STIM-Orai activating region (SOAR) of STIM1. STIM1 interacts with Ca(V)1.2 channels and localizes within discrete endoplasmic reticulum/plasma membrane junctions containing both Ca(V)1.2 and Orai1 channels. Hence, STIM1 interacts with and reciprocally controls two major calcium channels hitherto thought to operate independently. Such coordinated control of the widely expressed Ca(V)1.2 and Orai channels has major implications for Ca(2+) signal generation in excitable and nonexcitable cells.

STIM protein coupling in the activation of Orai channels.

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca(2+) changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca(2+) entry through the Orai family of highly Ca(2+)-selective "store-operated" channels (SOCs). Dissecting the STIM-Orai coupling process is restricted by the abstruse nature of the ER-PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca(2+) release-activated Ca(2+) (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct-Orai1 coupling occurred in STIM1/STIM2(-/-) DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct-Orai1 and S2-ct-Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB's action, the locked STIMct-Orai complex provides a potentially useful probe to structurally examine coupling.

Pentylenetetrazole-induced status epilepticus following training does not impair expression of morphine-induced conditioned place preference.

Learning and memory play an important role in morphine addiction. Status epilepticus (SE) can impair the spatial and emotional learning and memory. However, little is known about the effects of SE on morphine-induced conditioned place preference (CPP). The present study was designed to investigate the effects of SE on morphine CPP, with food CPP being used as a control. The effects of SE on spatial memory in the Morris water maze (MWM) and Y-maze were investigated. SE was induced in adult mice using intraperitoneal injection of pentylenetetrazole; control mice received saline. The data indicated that SE had no effects on the formation of morphine CPP; however, the formation of food CPP was blocked by SE. Meanwhile, spatial memory assayed in the MWM and Y-maze was impaired by SE. In addition, the data demonstrated that SE did not cause a lasting disturbance of motor activity nor a change in the mice's appetite. These results suggested that although SE had no effects on morphine CPP, there was impaired food CPP and spatial memory in both the MWM and the Y-maze. The mechanisms underlying memory process of morphine CPP may be different from other types of memory.

An andersen-Tawil syndrome mutation in Kir2.1 (V302M) alters the G-loop cytoplasmic K+ conduction pathway.

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.

Orai1 and STIM reconstitute store-operated calcium channel function.

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.